The aging endothelium is characterized by reduced activity of the endothelial dilator NO, increased production of ROS and oxidant stress, and increased inflammatory and thrombotic activity. Endothelial storage granules, including Weibel-Palade Bodies (WPBs) contain numerous mediators that play important roles in regulating vasomotor, thrombotic and inflammatory processes, and can be rapidly released by stimulated exocytosis. It is likely that exocytosis and release of these endothelial granules play an important role in the vascular pathology associated with aging. However, no previous studies have directly assessed the effects of aging on this fundamental mechanism. In novel preliminary results, we demonstrate that native endothelium of aging blood vessels have increased ability to generate and release pathophysiological mediators (including endothelin-1) during stimulated exocytosis. Our overall hypothesis is that increased release and generation of endothelial mediators by stimulated exocytosis contributes to vascular dysfunction in aging. We hope that by increasing our understanding of how aging affects this fundamental mechanism, it may provide novel approaches to treat diseases associated with aging. We will test our hypotheses on native endothelium of young and aging blood vessels, using the Fischer rat preclinical model of aging. Three specific aims are proposed to: AIM 1: DETERMINE THE INFLUENCE OF AGING ON THE GENERATION AND EXOCYTOSIS OF ENDOTHELIN-1 FROM ENDOTHELIUM. These experiments will focus on evaluating the increased ability of aging endothelium to generate endothelin-1 (ET-1) during exocytosis. Experiments will investigate the expression, processing and exocytotic release of ET-1 from the endothelium of young and aging blood vessels. AIM 2: DETERMINE THE ROLE OF NITRIC OXIDE IN REGULATING ENDOTHELIAL WPB EXOCYTOSIS IN AGING BLOOD VESSELS. These experiments will investigate the apparent decreased inhibitory effect of NO signaling in regulating exocytosis in aging endothelium. Experiments will focus on the mechanisms underlying NO regulation of exocytosis. AIM 3: EVALUATE THE PATHOLOGICAL ROLE OF ENDOTHELIAL EXOCYTOSIS IN THE AGING MICROCIRCULATION. These experiments will evaluate the potential pathophysiological role of dysregulated endothelial exocytosis in aging. This will be achieved by investigating the role of exocytosis in the microvascular responses of young and aging animals to ischemia- reperfusion of the mesenteric circulation. PUBLIC HEALTH RELEVANCE: The Johns Hopkins physician and icon of modern medicine, Sir William Osler (1849-1919) commented that we are only as old as our arteries. We now know that we may only be as old as our endothelium. This project proposes to analyze the influence of aging on a fundamental mechanism of endothelial function (stimulated exocytosis), and to evaluate its role in stress- induced microvascular dysfunction associated with aging organs.